The preparation of protein samples for analysis, for example, mass spectrometry analysis, typically includes three main steps: solubilization, digestion, and peptide recovery. Current methods and reagents applied to one step are rarely compatible with the following step. For example, solubilizing agents such as surfactants (e.g., sodium dodecyl sulfate (SDS)) or denaturants (e.g., acetonitrile, urea, or guanidine) typically inhibit the proteases, such as trypsin, that are used in the digestion that follows solubilization. Even when used in concentrations that can be tolerable for trypsin activity, the presence of these surfactants or denaturants interfere with subsequent analyses, such as liquid chromatography or mass spectrometric analysis. Accordingly, removal of the surfactants and organic solvents is typically required before conducting further analysis on a sample (e.g., proteins or peptides). The manipulations required for removal of these reagents complicates the sample preparation process and often leads to loss of sample material.
The digestion step frequently presents a major challenge in protein sample preparation. A typical protein digestion with trypsin requires overnight incubation to reach completion. Even after overnight incubation, some proteins that are resistant to digestion, such as membrane proteins, can remain intact, thus requiring extraordinary conditions to achieve satisfactory digestion. Current methods employed in an attempt to overcome these limitations and to speed the digestion process include the use of organic solvents (e.g., acetonitrile), elevated temperatures, denaturants (e.g., urea), and/or detergents (e.g., SDS) to improve protein solubilization and protein denaturation, thus improving digestion. However, these alternative methods and additives often result in incomplete cleavage and low reproducibility, limiting their utility. The use of these reagents also leads to inhibition of trypsin activity, interference with HPLC separation, and suppression of peptide detection in mass spectrometry.
In-gel protein digestion brings specific challenges to protein sample preparation. Success of in-gel digestion relies not only on efficient protein digestion but also on efficient post-digestion peptide extraction from the gel. Peptide extraction from the gel can be lengthy and laborious and it is often only moderately efficient in terms of peptide recovery. Recovered peptides are generally limited to the size of about 2,500 Da. Longer peptides are largely trapped in the gel. See “In-gel digestion with endoproteinase Lys-C”, Y. Wada, M. Kadoya, J. of Mass Spectrom. 2003; 38: 117-118. Recovery of peptides with increased hydrophobicity is also impacted.
Other procedures related to protein sample preparation include analysis of post-translational protein modifications. About 60% of all human proteins are glycosylated. Glycosylation was shown to play important role in many key cellular mechanisms. To analyze glycosylation, a glycan should be separated from a protein. This removal, referred to as deglycosylation, is performed by using glycosidases. Deglycosylation is frequently a time-consuming process. Reagents such as sodium dodecyl sulfate (SDS) can dramatically improve deglycosylation, potentially by providing better access to glycan attachment sites for the glycosidases. However, SDS interferes with downstream sample preparation steps, mass spectrometric analysis, and HPLC analysis.
Accordingly, there is a need for improved methods for protein sample preparation. There is also a need for methods or reagents that benefit one or more of the three major protein preparation steps: solubilization, digestion, and peptide recovery, in order to streamline the protein sample preparation process. Preferably, these methods or reagents would not lead to the inhibition of protease activity, and would not interfere with isolation and/or characterization techniques. There is a particular need to streamline in-gel digestion protocols and to improve recovery of peptides from gels. Finally, there is a need for improved methods of protein deglycosylation that do not interfere with downstream sample preparation and mass spectrometric analysis of proteins and glycans.